Circulating MicroRNA

Circulating miRNAs were extracted from 200 μl EDTA-plasma using the miRCURY^TM RNA Isolation Kit–Biofluids (Exiqon A/S, Vedbaek, Denmark), adding 1 μg MS2 carrier RNA to each sample prior to RNA extraction to ensure high RNA yield and reproducibility. Synthesis of complementary DNA (cDNA) was performed using the Universal cDNA Synthesis Kit II (Exiqon A/S), whereby RNA input was optimized to 4 μl of total RNA per 10 μl reaction volume as template in the cDNA synthesis reaction. Synthetic oligonucleotides were added to the plasma samples using the miRCURY^TM RNA Spike-in Kit (Exiqon A/S) according to the manufacturer’s protocol to monitor cDNA synthesis and PCR amplification.
The selection of the specific set of miRNAs included in these assays was guided by their usefulness as biomarkers for sample quality, their stable expression in plasma, and their independence from status disease. The miRCURY^TM LNA (Exiqon A/S) miRNA profiling technology was used for two purposes: firstly to assess the robustness and quality of the RNA isolation, cDNA synthesis and overall sample quality, and secondly, to include miRNAs of interest in epidemiological research. A total of nine miRNAs were included in two qPCR panels; the full nomenclature, accession numbers and sequences for the nine miRNAs were retrieved from miRbase, release 22 [5 (link)] and are shown in S1 Table. The first qPCR panel used was the QC PCR Panel V4.M; sample quality was assessed by examining adding synthetic oligonucleotides, as well as six miRNAs expected to be highly abundant in specific sets of samples: miR-30c-5p, expressed in cerebrospinal fluid; miR-103a-3p and miR-191-5p, expressed in most tissues; miR-124-3p, expressed in kidney and urine samples; and miR-451a and miR-23a-3p, indicators of hemolysis and internal controls that are well detected in plasma and serum [35 (link), 39 (link)]. The second qPCR panel featured four miRNAs (miR-93-5p, miR-24-3p, miR-23a-3p, and miR-33b-5p); the first three miRNAs of this panel were included in this experiment because of their stable expression in plasma samples [40 (link)–42 ], whereas miR-33b-5p is of epidemiological interest in the KORA survey because the methylation level of its encoding region has been found to be associated with lipid levels [43 (link)].
All qPCR assays were conducted using the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) following the specific recommendations for ABI instruments and the manufacturer’s instructions for each qPCR panel, e.g. number of amplification cycles (45 for both panels in order to maximize comparability between them)Data was processed using the SDS 4.2 software (Applied Biosystems). This miRNA profiling platform is sensitive and specific, as shown by the appropriate assay-specific melting temperatures in the melting curve analyses from our data (S1 Fig) and the platform’s performance in comparison to other detection systems in terms of assay cross-reactivity and specificity [24 (link)]. For all analyses in this study, miRNA expression was quantified as Cq, the PCR cycle at which the target is detected, as defined by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [44 (link)].

SAR trial. The simulated SAR trial required dogs to find a target odor (a hidden operator and his breath, simulating a missing person) on a rubble field (30 × 35 m), within a maximum time of 15 min. Blood was collected at T0 immediately before the SAR (resting baseline) and at T1 later in the SAR (physiological stress). T0 versus T1 enables a relation between two different physiological states, where T1 comprises a multifactorial combination of stresses physiologically recovered in trained dogs [15 (link)].
The experimental framework of the study is divided into two phases.

In the first step, dog blood was drawn at T0 (n = 22) and T1 (n = 22) (dogs reported in Table 1), and total serum RNA was isolated. The total RNA of six dogs listed in Table 1 (dog ID: A, B, C, D, E, and F) was used for library preparation and NGS miRNome sequencing. This step aimed to evaluate the T0 versus T1 unregulated circulating miRNAs, i.e., candidate stable EC miRNAs.

In the second step, unregulated cmiRNAs selected as candidate EC were validated by qPCR in a larger panel of dogs, including all dogs listed in Table 1 (T0 n = 22; T1 n = 22). Then, bioinformatic analysis of the qPCR results identified the EC miRNA stability values and the EC final ranking (Figure 1).

This study was approved by the University of California, San Francisco (UCSF) Institutional Review Board (18–26351). Patients ≥ 55 years of age were recruited at the UCSF Vascular Surgery outpatient clinics and vascular laboratories between January 2019 and 2020. All patients who underwent screening or surveillance imaging for AAA during this time were reviewed for possible study participation based on the inclusion and exclusion criteria stated below [4 (link)]. Cases were defined as patients with a maximum infrarenal abdominal aorta diameter of ≥ 3 cm on US or computed tomography (CT). Controls, or patients without aneurysms, were defined as having a maximum infrarenal abdominal aorta diameter of < 3 cm on US or CT. Patients were excluded from participating if they had severe hepatic (Child-Pugh ≥ B) or renal (creatinine ≥ 2 mg/dL) dysfunction, a non-vascular inflammatory disease, a severe acute illness or surgery within 30 days, or were taking immunosuppressive medications or steroids (Table 1). The inclusion and exclusion criteria were selected with the goal of reducing the possible influence of acute and chronic diseases that may directly affect circulating miRNA patterns, with a focus on diseases associated with changes in systemic inflammation.